Etiologic Risk Group (see link below): Risk Group 2 Parasitic Agent
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmaniasis is a parasitic disease that is found in parts of the tropics, subtropics, and southern Europe. Leishmaniasis is caused by infection with Leishmania parasites, which are spread by the bite of infected sand flies. Some people have a silent infection, without any symptoms or signs. People who develop clinical evidence of infection have one or more sores on their skin. The sores can change in size and appearance over time. The sores may start out as papules (bumps) or nodules (lumps) and may end up as ulcers (like a volcano, with a raised edge and central crater); skin ulcers may be covered by scab or crust. The sores usually are painless but can be painful. Some people have swollen glands near the sores (for example, under the arm, if the sores are on the arm or hand).
Essentiality of this protein:
Malate dehydrogenase (EC:1.1.1.37) (MDH) [PMID: 1610875] catalyzes the interconversion of malate to oxaloacetate utilizing the NAD/NADH cofactor system. The enzyme participates in the citric acid cycle and exists in all aerobics organisms. While prokaryotic organisms contains a single form of MDH, in eukaryotic cells there are two isozymes: one which is located in the mitochondrial matrix and the other in the cytoplasm. Fungi and plants also harbor a glyoxysomal form which functions in the glyoxylate pathway. In plants chloroplast there is an additional NADP-dependent form of MDH (EC:1.1.1.82) which is essential for both the universal C3 photosynthesis (Calvin) cycle and the more specialised C4 cycle. The pattern for this enzyme includes two residues involved in the catalytic mechanism [PMID: 2775751]: an aspartic acid which is involved in a proton relay mechanism, and an arginine which binds the substrate. http://www.ebi.ac.uk/interpro/entry/IPR001252?q=malate%20dehydrogenase%20type:active_site
Complex of proteins?: Yes Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Gene/Ortholog: mtu1261 (OG4_10831); Phenotype: significant loss of fitness; Source study: nmpdr
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
One unit will convert 1.0 μmole of oxalacetate and β-NADH to L-malate and β-NAD per min at pH 7.5 at 25 °C. -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information http://www.sciencedirect.com/science/article/pii/S0166685106001411 -- links to assay reagents (substrates) pages. http://www.sigmaaldrich.com/catalog/product/sigma/m7032?lang=en®ion=US --- List cost and quantity of substrate reagents, supplier, and catalog #
Lyophilized powder, 50units/mg solid $39.00 USD, Catalog M7032
Structure Available (PDB or Homology model)
Figure 2. Crystal structure of a putative Cytosolic malate dehydrogenase from Leishmania major Friedlin. Protein chains are colored from the N-terminal to the C-terminal using a rainbow (spectral) color gradient.
-- PDB # or closest PDB entry if using homology model: 4H7P
-- For Homology Model option: NO SIGNIFICANT SIMILARITY FOUND
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: [(4-{2-[(5-hydroxy-2-{[(4-methoxyphenyl)methyl]carbamoyl}-4-oxo-4H-chromen-7-yl)oxy]ethyl}phenyl)carbamoyl]formic acid 7-[8-formyl-1,6,7-trihydroxy-3-methyl-5-(propan-2-yl)naphthalen-2-yl]-2,3,8-trihydroxy-6-methyl-4-(propan-2-yl)naphthalene-1-carbaldehyde (+-)-Econazole::1-(2,4-Dichloro-beta-((p-chlorobenzyl)oxy)phenethyl)imidazole::1-{2-(4-chlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl}-1H-imidazole 3-(4,5-Diphenyl-oxazol-2-yl)-propionic acid
Expression Information (has it been expressed in bacterial cells):
Malate dehydrogenase (mdh) gene expression in Escherichia coli in response to oxygen, carbon, and heme availability. http://www.ncbi.nlm.nih.gov/pubmed/7592446
Image of protein (PyMol with features delineated and shown separately):
Figure 3. Image of protein 4H7P shown as sticks having carbons colored green, oxygens colored red, and nitrogens colored blue.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): CGACAGAAGGCCACAAACTTCGAGGACGAACGAAGGCAACAACAGCGGGACAGTGTGTGTGTCTGTGTGT GTGTCTGTGTCACCTTCCCCCCCCCCACTCCTATCACTTCTCTTCTTTTTTTTTCTGCACCTCTCACACT GCGCAATGTCCGCTGTGAAGGTGGCCGTGACGGGTGCGGCTGGTCAGATCGGGTATGCATTGGTACCGCT GATCGCGCGCGGCGCCCTCCTCGGGCCCACCACGCCTGTGGAGCTCCGCCTGCTGGATATCGAGCCCGCT CTGAAGGCGCTTGCCGGCGTCGAAGCGGAGCTGGAAGACTGCGCCTTCCCTCTCCTGGACAAGGTCGTCG TCACGGCAGACCCGCGCGTTGCCTTCGACGGGGTCGCCATTGCCATCATGTGCGGTGCCTTCCCGCGCAA GGCAGGGATGGAGCGCAAGGACCTCCTCGAGATGAACGCGCGCATCTTCAAGGAGCAGGGTGAGGCCATC GCTGCTGTGGCCGCGTCAGACTGCCGCGTGGTGGTTGTCGGCAATCCGGCCAACACAAACGCTCTCATCC TCCTCAAGTCAGCTCAGGGGAAGTTGAACCCCCGTCATGTGACCGCCATGACCCGCCTCGATCACAACCG TGCCTTGTCTCTTCTGGCTCGCAAGGCCGGTGTGCCGGTGTCGCAGGTGCGCAACGTGATCATCTGGGGC AACCATAGCTCTACACAGGTCCCTGACACTGACAGCGCTGTGATCGGCACCACTCCGGCCCGCGAAGCGA TCAAGGACGATGCACTCGATGACGACTTTGTGCAGGTGGTGCGCGGGCGCGGCGCCGAAATCATCCAACT ACGTGGTCTTTCCTCAGCCATGTCCGCCGCCAAGGCCGCCGTAGACCACGTACATGACTGGATACATGGC ACACCGGAGGGCGTGTACGTTTCGATGGGCGTGTACTCGGATGAAAACCCGTACGGCGTGCCGAGCGGCC TCATCTTCTCCTTTCCGTGCACGTGCCACGCCGGCGAGTGGACTGTTGTCTCGGGCAAGCTGAATGGGGA CCTCGGGAAGCAGCGTCTCGCCAGCACGATCGCCGAGCTGCAGGAGGAGAGGGCACAGGCAGGGCTATGA GCCAAGCATCGCTGCACTCATGTCAAGAGGTTGTGCGTTGGCCCTCTTGCAGTTCAGTAGCGCTCGCGGT GCCGCCGCCGTCCCCCCCCCCTTCTAGTATATGTGCGAGCGTGGGGAAGGGGAGGGGAGGGGGTCGGGAA AAAACAT
*length of your protein in Amino Acids 345
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 73446.29 (Da)
Molar Extinction coefficient of your protein at 280 nm wavelength: Coefficient is 47520 M-1cm-1 measured at 280nm in water.
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
*NCBI Gene # or RefSeq#: 5653501
*Protein ID (NP or XP #) or Wolbachia#: RU003405
*Organism (including strain): Leishmana major, Friedlin strain
Etiologic Risk Group (see link below): Risk Group 2 Parasitic Agent
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmaniasis is a parasitic disease that is found in parts of the tropics, subtropics, and southern Europe. Leishmaniasis is caused by infection with Leishmania parasites, which are spread by the bite of infected sand flies. Some people have a silent infection, without any symptoms or signs. People who develop clinical evidence of infection have one or more sores on their skin. The sores can change in size and appearance over time. The sores may start out as papules (bumps) or nodules (lumps) and may end up as ulcers (like a volcano, with a raised edge and central crater); skin ulcers may be covered by scab or crust. The sores usually are painless but can be painful. Some people have swollen glands near the sores (for example, under the arm, if the sores are on the arm or hand).
Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=22073
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/gene/?term=cMDH+LMJF_28_2860
Essentiality of this protein:
Malate dehydrogenase (EC:1.1.1.37) (MDH) [PMID: 1610875] catalyzes the interconversion of malate to oxaloacetate utilizing the NAD/NADH cofactor system. The enzyme participates in the citric acid cycle and exists in all aerobics organisms. While prokaryotic organisms contains a single form of MDH, in eukaryotic cells there are two isozymes: one which is located in the mitochondrial matrix and the other in the cytoplasm. Fungi and plants also harbor a glyoxysomal form which functions in the glyoxylate pathway. In plants chloroplast there is an additional NADP-dependent form of MDH (EC:1.1.1.82) which is essential for both the universal C3 photosynthesis (Calvin) cycle and the more specialised C4 cycle. The pattern for this enzyme includes two residues involved in the catalytic mechanism [PMID: 2775751]: an aspartic acid which is involved in a proton relay mechanism, and an arginine which binds the substrate. http://www.ebi.ac.uk/interpro/entry/IPR001252?q=malate%20dehydrogenase%20type:active_site
Complex of proteins?: Yes
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Gene/Ortholog: mtu1261 (OG4_10831); Phenotype: significant loss of fitness; Source study: nmpdr
*EC#: 1.1.1.37
Link to BRENDA EC# page: http://brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.37
-- Show screenshot of BRENDA enzyme mechanism schematic
Figure 1. BRENDA enzyme mechanism schematic for the reaction catalyzed by malate dehydrogenase.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
One unit will convert 1.0 μmole of oxalacetate and β-NADH to L-malate and β-NAD per min at pH 7.5 at 25 °C.
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
http://www.sciencedirect.com/science/article/pii/S0166685106001411
-- links to assay reagents (substrates) pages.
http://www.sigmaaldrich.com/catalog/product/sigma/m7032?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
Lyophilized powder, 50units/mg solid $39.00 USD, Catalog M7032
Structure Available (PDB or Homology model)
Figure 2. Crystal structure of a putative Cytosolic malate dehydrogenase from Leishmania major Friedlin. Protein chains are colored from the N-terminal to the C-terminal using a rainbow (spectral) color gradient.
-- PDB # or closest PDB entry if using homology model: 4H7P
-- For Homology Model option: NO SIGNIFICANT SIMILARITY FOUND
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors:
[(4-{2-[(5-hydroxy-2-{[(4-methoxyphenyl)methyl]carbamoyl}-4-oxo-4H-chromen-7-yl)oxy]ethyl}phenyl)carbamoyl]formic acid
7-[8-formyl-1,6,7-trihydroxy-3-methyl-5-(propan-2-yl)naphthalen-2-yl]-2,3,8-trihydroxy-6-methyl-4-(propan-2-yl)naphthalene-1-carbaldehyde
(+-)-Econazole::1-(2,4-Dichloro-beta-((p-chlorobenzyl)oxy)phenethyl)imidazole::1-{2-(4-chlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl}-1H-imidazole
3-(4,5-Diphenyl-oxazol-2-yl)-propionic acid
Expression Information (has it been expressed in bacterial cells):
Malate dehydrogenase (mdh) gene expression in Escherichia coli in response to oxygen, carbon, and heme availability. http://www.ncbi.nlm.nih.gov/pubmed/7592446
Image of protein (PyMol with features delineated and shown separately):
Figure 3. Image of protein 4H7P shown as sticks having carbons colored green, oxygens colored red, and nitrogens colored blue.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
CGACAGAAGGCCACAAACTTCGAGGACGAACGAAGGCAACAACAGCGGGACAGTGTGTGTGTCTGTGTGT GTGTCTGTGTCACCTTCCCCCCCCCCACTCCTATCACTTCTCTTCTTTTTTTTTCTGCACCTCTCACACT GCGCAATGTCCGCTGTGAAGGTGGCCGTGACGGGTGCGGCTGGTCAGATCGGGTATGCATTGGTACCGCT GATCGCGCGCGGCGCCCTCCTCGGGCCCACCACGCCTGTGGAGCTCCGCCTGCTGGATATCGAGCCCGCT CTGAAGGCGCTTGCCGGCGTCGAAGCGGAGCTGGAAGACTGCGCCTTCCCTCTCCTGGACAAGGTCGTCG TCACGGCAGACCCGCGCGTTGCCTTCGACGGGGTCGCCATTGCCATCATGTGCGGTGCCTTCCCGCGCAA GGCAGGGATGGAGCGCAAGGACCTCCTCGAGATGAACGCGCGCATCTTCAAGGAGCAGGGTGAGGCCATC GCTGCTGTGGCCGCGTCAGACTGCCGCGTGGTGGTTGTCGGCAATCCGGCCAACACAAACGCTCTCATCC TCCTCAAGTCAGCTCAGGGGAAGTTGAACCCCCGTCATGTGACCGCCATGACCCGCCTCGATCACAACCG TGCCTTGTCTCTTCTGGCTCGCAAGGCCGGTGTGCCGGTGTCGCAGGTGCGCAACGTGATCATCTGGGGC AACCATAGCTCTACACAGGTCCCTGACACTGACAGCGCTGTGATCGGCACCACTCCGGCCCGCGAAGCGA TCAAGGACGATGCACTCGATGACGACTTTGTGCAGGTGGTGCGCGGGCGCGGCGCCGAAATCATCCAACT ACGTGGTCTTTCCTCAGCCATGTCCGCCGCCAAGGCCGCCGTAGACCACGTACATGACTGGATACATGGC ACACCGGAGGGCGTGTACGTTTCGATGGGCGTGTACTCGGATGAAAACCCGTACGGCGTGCCGAGCGGCC TCATCTTCTCCTTTCCGTGCACGTGCCACGCCGGCGAGTGGACTGTTGTCTCGGGCAAGCTGAATGGGGA CCTCGGGAAGCAGCGTCTCGCCAGCACGATCGCCGAGCTGCAGGAGGAGAGGGCACAGGCAGGGCTATGA GCCAAGCATCGCTGCACTCATGTCAAGAGGTTGTGCGTTGGCCCTCTTGCAGTTCAGTAGCGCTCGCGGT GCCGCCGCCGTCCCCCCCCCCTTCTAGTATATGTGCGAGCGTGGGGAAGGGGAGGGGAGGGGGTCGGGAA AAAACAT
*length of your protein in Amino Acids 345
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 73446.29 (Da)
Molar Extinction coefficient of your protein at 280 nm wavelength: Coefficient is 47520 M-1cm-1 measured at 280nm in water.
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 4. TMpred graph of Malate Dehydrogenase of the Leishmania Major, protein 4H7P.
*CDS Gene Sequence (paste as text only):
ATGTCCGCTGTGAAGGTGGCCGTGACGGGTGCGGCTGGTCAGATCGGGTATGCATTGGTACCGCTGATCG CGCGCGGCGCCCTCCTCGGGCCCACCACGCCTGTGGAGCTCCGCCTGCTGGATATCGAGCCCGCTCTGAA GGCGCTTGCCGGCGTCGAAGCGGAGCTGGAAGACTGCGCCTTCCCTCTCCTGGACAAGGTCGTCGTCACG GCAGACCCGCGCGTTGCCTTCGACGGGGTCGCCATTGCCATCATGTGCGGTGCCTTCCCGCGCAAGGCAG GGATGGAGCGCAAGGACCTCCTCGAGATGAACGCGCGCATCTTCAAGGAGCAGGGTGAGGCCATCGCTGC TGTGGCCGCGTCAGACTGCCGCGTGGTGGTTGTCGGCAATCCGGCCAACACAAACGCTCTCATCCTCCTC AAGTCAGCTCAGGGGAAGTTGAACCCCCGTCATGTGACCGCCATGACCCGCCTCGATCACAACCGTGCCT TGTCTCTTCTGGCTCGCAAGGCCGGTGTGCCGGTGTCGCAGGTGCGCAACGTGATCATCTGGGGCAACCA TAGCTCTACACAGGTCCCTGACACTGACAGCGCTGTGATCGGCACCACTCCGGCCCGCGAAGCGATCAAG GACGATGCACTCGATGACGACTTTGTGCAGGTGGTGCGCGGGCGCGGCGCCGAAATCATCCAACTACGTG GTCTTTCCTCAGCCATGTCCGCCGCCAAGGCCGCCGTAGACCACGTACATGACTGGATACATGGCACACC GGAGGGCGTGTACGTTTCGATGGGCGTGTACTCGGATGAAAACCCGTACGGCGTGCCGAGCGGCCTCATC TTCTCCTTTCCGTGCACGTGCCACGCCGGCGAGTGGACTGTTGTCTCGGGCAAGCTGAATGGGGACCTCG GGAAGCAGCGTCTCGCCAGCACGATCGCCGAGCTGCAGGAGGAGAGGGCACAGGCAGGGCTATGA
*GC% Content for gene: 71.42%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**